Antibodies to calcium independent cytosolic phospholipase A2/B enzymes

ABSTRACT

The invention provides a novel calcium-independent cytosolic phospholipase A 2 /B enzyme, polynucleotides encoding such enzyme, antibodies to such enzyme, and methods for screening unknown compounds for anti-inflammatory activity mediated by the arachidonic acid cascade.

This application is a divisional of U.S. application Ser. No.09/927,180, filed Aug. 9, 2001, now U.S. Pat. No. 6,645,736, which is acontinuation of application Ser. No. 09/519,223, filed Mar. 6, 2000, nowU.S. Pat. No. 6,274,140, which is a continuation of application Ser. No.09/149,988, filed Sep. 9, 1998, now abandoned, which is a divisional ofapplication Ser. No. 08/555,568, filed Nov. 8, 1995, now U.S. Pat. No.5,976,854, which is a continuation-in-part of application Ser. No.08/281,193, filed Jul. 27, 1994, now U.S. Pat. No. 5,466,595, which is acontinuation-in-part of application Ser. No. 08/422,106, filed Apr. 14,1995, now U.S. Pat. No. 5,589,170, which is a continuation-in-part ofapplication Ser. No. 08/422,420, filed Apr. 14, 1995, now U.S. Pat. No.5,554,511. This application also claims benefit of PCT/US95/08069, filedJun. 26, 1995. U.S. application Ser. No. 08/555,568 is being relied uponand is incorporated herein by reference.

The present invention relates to a purified calcium independentcytosolic phospholipase A₂/B enzymes which are useful for assayingchemical agents for anti-inflammatory activity.

BACKGROUND OF THE INVENTION

The phospholipase A₂ enzymes comprise a widely distributed family ofenzymes which catalyze the hydrolysis of the acyl ester bond ofglycerophospholipids at the sn-2 position. One kind of phospholipase A₂enzymes, secreted phospholipase A₂ or sPLA₂, are involved in a number ofbiological functions, including phospholipid digestion, the toxicactivities of numerous venoms, and potential antibacterial activities. Asecond kind of phospholipase A₂ enzymes, the intracellular phospholipaseA₂ enzymes, also known as cytosolic phospholipase A₂ or cPLA₂, areactive in membrane phospholipid turnover and in regulation ofintracellular signalling mediated by the multiple components of thewell-known arachidonic acid cascade. One or more cPLA₂ enzymes arebelieved to be responsible for the rate limiting step in the arachidonicacid cascade, namely, release of arachidonic acid from membraneglycerophospholipids. The action of cPLA₂ also results in biosynthesisof platelet activating factor (PAF).

The phospholipase B enzymes are a family of enzymes which catalyze thehydrolysis of the acyl ester bond of glycerophospholipids at the sn-1and sn-2 positions. The mechanism of hydrolysis is unclear but mayconsist of initial hydrolysis of the sn-2 fatty acid followed by rapidcleavage of the sn-1 substituent, i.e., functionally equivalent to thecombination of phospholipase A₂ and lysophospholipase (Saito et al.,Methods of Enzymol., 1991, 197, 446; Gassama-Diagne et al., J. Biol.Chem., 1989, 264, 9470). Whether these two events occur at the same ortwo distinct active sites has not been resolved. It is also unknown ifthese enzymes have a preference for the removal of unsaturated fattyacids, in particular arachidonic acid, at the sn-2 position andaccordingly contribute to the arachidonic acid cascade.

Upon release from the membrane, arachidonic acid may be metabolized viathe cyclooxygenase pathway to produce the various prostaglandins andthromboxanes, or via the lipoxygenase pathway to produce the variousleukotrienes and related compounds. The prostaglandins, leukotrienes andplatelet activating factor are well known mediators of variousinflammatory states, and numerous anti-inflammatory drugs have beendeveloped which function by inhibiting one or more steps in thearachidonic acid cascade. Use of the present anti-inflammatory drugswhich act through inhibition of arachidonic acid cascade steps has beenlimited by the existence of side effects which may be harmful to variousindividuals.

A very large industrial effort has been made to identify additionalanti-inflammatory drugs which inhibit the arachidonic acid cascade. Ingeneral, this industrial effort has employed the secreted phospholipaseA₂ enzymes in inhibitor screening assays, for example, as disclosed inU.S. Pat. No. 4,917,826. However, because the secreted phospholipase A₂enzymes are extracellular proteins (i.e., not cytosolic) and are notspecific for hydrolysis of arachidonic acid, they are presently notbelieved to participate directly in the arachidonic acid cascade. Whilesome inhibitors of the small secreted phospholipase A₂ enzymes haveanti-inflammatory action, such as indomethacin, bromphenacyl bromide,mepacrine, and certain butyrophenones as disclosed in U.S. Pat. No.4,239,780, it is presently believed that inhibitor screening assaysshould employ cytosolic phospholipase A₂ enzymes which directlyparticipate in the arachidonic acid cascade.

An improvement in the search for anti-inflammatory drugs which inhibitthe arachidonic acid cascade was developed in commonly assigned U.S.Pat. No. 5,322,776, incorporated herein by reference. In thatapplication, a cytosolic form of phospholipase A₂ was identified,isolated, and cloned. Use of the cytosolic form of phospholipase A₂ toscreen for anti-inflammatory drugs provides a significant improvement inidentifying inhibitors of the arachidonic acid cascade. The cytosolicphospholipase A₂ disclosed in U.S. Pat. No. 5,322,776 is a 110 kDprotein which depends on the presence of elevated levels of calciuminside the cell for its activity. The cPLA₂ of U.S. Pat. No. 5,322,776plays a pivotal role in the production of leukotrienes andprostaglandins initiated by the action of pro-inflammatory cytokines andcalcium mobilizing agents. The cPLA₂ of U.S. Pat. No. 5,322,776 isactivated by phosphorylation on serine residues and increasing levels ofintracellular calcium, resulting in translocation of the enzyme from thecytosol to the membrane where arachidonic acid is selectively hydrolyzedfrom membrane phospholipids.

In addition to the cPLA₂ of U.S. Pat. No. 5,322,776, some cells containcalcium independent phospholipase A₂/B enzymes. For example, suchenzymes have been identified in rat, rabbit, canine and human hearttissue (Gross, TCM, 1991, 2, 115; Zupan et al., J. Med. Chem., 1993, 36,95; Hazen et al., J. Clin. Invest., 1993, 91, 2513; Lehman et al., J.Biol. Chem., 1993, 268, 20713; Zupan et al., J: Biol. Chem., 1992, 267,8707; Hazen et al., J. Biol. Chem., 1991, 266, 14526; Loeb et al., J.Biol. Chem., 1986, 261, 10467; Wolf et al., J. Biol. Chem., 1985, 260,7295; Hazen et al., Meth. Enzymol., 1991, 197, 400; Hazen et al., J.Biol. Chem., 1990, 265, 10622; Hazen et al., J. Biol. Chem., 1993, 268,9892; Ford et al., J. Clin. Invest., 1991, 88, 331; Hazen et al., J.Biol. Chem., 1991, 266, 5629; Hazen et al., Circulation Res., 1992, 70,486; Hazen et al., J. Biol. Chem., 1991, 266, 7227; Zupan et al., FEBS,1991, 284, 27), as well as rat and human pancreatic islet cells(Ramanadham et al., Biochemistry, 1993, 32, 337; Gross et al.,Biochemistry, 1993, 32, 327), in the macrophage-like cell line, P388D₁(Ulevitch et al., J. Biol. Chem., 1988, 263, 3079; Ackermann et al., J.Biol. Chem., 1994, 269, 9227; Ross et al., Arch. Biochem. Biophys.,1985, 238, 247; Ackermann et al., FASEB Journal, 1993, 7(7), 1237), invarious rat tissue cytosols (Nijssen et al., Biochim. Biophys. Acta,1986, 876, 611; Pierik et al., Biochim. Biophys. Acta, 1988, 962, 345;Aarsman et al., J. Biol. Chem., 1989, 264, 10008), bovine brain (Ueda etal., Biochem. Biophys, Res. Comm., 1993, 195, 1272; Hirashima et al., J.Neurochem., 1992, 59, 708), in yeast (Saccharomyces cerevisiae)mitochondria (Yost et al., Biochem. International, 1991, 24, 199),hamster heart cytosol (Cao et al., J. Biol. Chem., 1987, 262, 16027),rabbit lung microsomes (Angle et al., Biochim. Biophys. Acta, 1988, 962,234) and guinea pig intestinal brush-border membrane (Gassama-Diagne etal., J. Biol. Chem., 1989, 264, 9470).

It is believed that the calcium independent phospholipase A₂/B enzymesmay perform important functions in release of arachidonic acid inspecific tissues which are characterized by unique membranephospholipids, by generating lysophospholipid species which aredeleterious to membrane integrity or by remodeling of unsaturatedspecies of membrane phospholipids through deacylation/reacylationmechanisms. The activity of such a phospholipase may well be regulatedby mechanisms that are different from that of the cPLA₂ of U.S. Pat. No.5,322,776. In addition the activity may be more predominant in certaininflamed tissues over others. Although the enzymatic activity is notdependent on calcium this does not preclude a requirement for calcium invivo, where the activity may be regulated by the interaction of otherprotein(s) whose function is dependent upon a calcium flux.

SUMMARY OF THE INVENTION

In certain embodiments, the present invention provides compositionscomprising a purified phospholipase enzyme characterized by (a) activityin the absence of calcium; (b) a molecular weight of 86 kD on SDS-PAGE;and (c) the presence of one or more amino acid sequences selected fromthe group consisting of NPHSGFR (SEQ ID NO:3), XASXGLNQVNK (SEQ ID NO:4)(X is preferably N or A), YGASPLHXAK (SEQ ID NO:5) (X is preferably W),DNMEMIK (SEQ ID NO:6), GVYFR (SEQ ID NO:7), MKDEVFR (SEQ ID NO:8),EFGEHTK (SEQ ID NO:9), VMLTGTLSDR (SEQ ID NO:10), XYDAPEVIR (SEQ IDNO:11) (X is preferably N), FNQNINLKPPTQPA (SEQ ID NO:12), XXGAAPTYFRP(SEQ ID NO:13) (X is preferably S), TVFGAK (SEQ ID NO:14), andXWSEMVGIQYFR (SEQ ID NO:15) (X is preferably A), wherein X representsany amino acid residue.

In other embodiments, the invention provides compositions comprising apurified phospholipase enzyme characterized by (a) activity in theabsence of calcium; (b) a molecular weight of 86 kD on SDS-PAGE; and (c)the presence of one or more amino acid sequences selected from the groupconsisting of YGASPLHXAK, MKDEVFR, EFGEHTK, VMLTGTLSDR, XXGAAPTYFRP andTVFGAK, wherein X represents any amino acid residue.

Certain embodiments provide compositions comprising a purified mammaliancalcium independent phospholipase A₂/B enzyme.

In other embodiments, the enzyme is further characterized by activity ina mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine (preferably aspecific activity of about 1 μmol to about 20 μmol per minute permilligram, more preferably a specific activity of about 1 μmol to about5 μmol per minute per milligram); by a pH optimum of 6; and/or by theabsence of stimulation by adenosine triphosphate in the liposome assay.

In other embodiments, the invention provides isolated polynucleotidescomprising a nucleotide sequence selected from the group consisting of:(a) the nucleotide sequence of SEQ ID NO:1; (b) a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO:2; (c) a nucleotidesequence encoding a fragment of the amino acid sequence of SEQ ID NO:2having activity in a mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (d) a nucleotidesequence capable of hybridizing with the sequence of (a), (b) or (c)which encodes a peptide having activity in a mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; and (e) allelicvariants of the sequence of (a). Other embodiments provide an isolatedpolynucleotide comprising a nucleotide sequence selected from the groupconsisting of: (a) the nucleotide sequence of SEQ ID NO:16; (b) anucleotide sequence encoding the amino acid sequence of SEQ ID NO:17;(c) a nucleotide sequence encoding a fragment of the amino acid sequenceof SEQ ID NO:17 having activity in a mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (d) the nucleotidesequence of SEQ ID NO: 18; (e) a nucleotide sequence encoding the aminoacid sequence of SEQ ID NO: 19; (f) a nucleotide sequence encoding afragment of the amino acid sequence of SEQ ID NO: 19 having activity ina mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (g) the nucleotidesequence of SEQ ID NO:20; (h) a nucleotide sequence encoding the aminoacid sequence of SEQ ID NO:21; (i) a nucleotide sequence encoding afragment of the amino acid sequence of SEQ ID NO:21 having activity in amixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (j) the nucleotidesequence of SEQ ID NO:22; (l) a nucleotide sequence encoding the aminoacid sequence of SEQ ID NO:23; (1) a nucleotide sequence encoding afragment of the amino acid sequence of SEQ ID NO:23 having activity in amixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (m) a nucleotidesequence capable of hybridizing with the sequence of any of (a)-(l)which encodes a peptide having activity in a mixed mice le assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; and (n) allelicvariants of the sequence of (a), (d), (g) or (j). Expression vectorscomprising such polynucleotides and host cells transformed with suchvectors are also provided by the present invention. Compositionscomprising peptides encoded by such polynucleotides are also provided.

The present invention also provides processes for producing aphospholipase enzyme, said process comprising: (a) establishing aculture of the host cell transformed with a cPLA₂/B encodingpolynucleotide in a suitable culture medium; and (b) isolating saidenzyme from said culture. Compositions comprising a peptide madeaccording to such processes are also provided.

Certain embodiments of the present invention provide compositionscomprising a peptide comprising an amino acid sequence selected from thegroup consisting of: (a) the amino acid sequence of SEQ ID NO:2; and (b)a fragment of the amino acid sequence of SEQ ID NO:2 having activity ina mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine.

Other embodiments provide compositions comprising a peptide comprisingan amino acid sequence selected from the group consisting of: (a) theamino acid sequence of SEQ ID NO:17; (b) a fragment of the amino acidsequence of SEQ ID NO:17 having activity in a mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (c) the amino acidsequence of SEQ ID NO:19; (d) a fragment of the amino acid sequence ofSEQ ID NO:19 having activity in a mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (e) the amino acidsequence of SEQ ID NO:21; (f) a fragment of the amino acid sequence ofSEQ ID NO:21 having activity in a mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine; (g) the amino acidsequence of SEQ ID NO:23; and (h) a fragment of the amino acid sequenceof SEQ ID NO:23 having activity in a mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine.

The present invention also provides methods for identifying an inhibitorof phospholipase activity, said method comprising: (a) combining aphospholipid, a candidate inhibitor compound, and a compositioncomprising a phospholipase enzyme peptide; and (b) observing whethersaid phospholipase enzyme peptide cleaves said phospholipid and releasesfatty acid thereby, wherein the peptide composition is one of thosedescribed above. Inhibitor of phospholipase activity identified by suchmethods, pharmaceutical compositions comprising a therapeuticallyeffective amount of such inhibitors and a pharmaceutically acceptablecarrier, and methods of reducing inflammation by administering suchpharmaceutical compositions to a mammalian subject are also provided.

Polyclonal and monoclonal antibodies to the peptides of the inventionare also provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Fractions containing activity eluted from a Mono P column wereexamined by reducing SDS-PAGE on a 4–20% gradient gel. Activity of eachfraction is show above the gel and the 86 kD band is indicated on thesilver stained gel. Molecular weight markers are indicated.

FIG. 2: Active fractions from a Mono p/Heparin column were combined andfurther purified on a size exclusion column. Activity eluted in the250–350 kD size range. Examination of the fractions by SDS-PAGE underreducing conditions on 4–20% gel indicated only one protein bandcorrelated with activity at 86 kD. Molecular weight markers areindicated.

FIG. 3: Active fractions from Mono P eluate and cPLA₂ (0.1–1.0 μg) wereanalyzed on two 4–20% SDS gels under reducing conditions run inparallel. One gel was silver stained (A) and in the other gel theproteins were transferred to nitrocellulose. the blot was than probedwith an anti-cPLA, polyclonal antibody and reactive proteins werevisualized with the ECL system (Amersham) (B). Molecular weight markersare indicated.

FIG. 4: The activity of the calcium-independent phospholipase elutedfrom a Mono P/Heparin column and cPLA₂ were compared under conditionswhich favor each enzyme; pH 7, 10% glycerol in the absence of calciumand pH 9, 70% glycerol in the presence of calcium, respectively.

FIG. 5: Activity in the cytosolic extracts of COS cells transfectedwith: no DNA; plasmid (pED) containing no inserted gene; clone 9 in theantisense orientation; and clones 49, 31 and 9 expressed in pED. Theextracts were analyzed under two different assay conditions describedfor the data presented in FIG. 4.

FIG. 6: A comparison of sn-2 fatty acid hydrolysis by activity elutedfrom a Mono P/Heparin column as a function of the fatty acid substituentat either the sn-1 or sn-2 position and the head group. HAPC, SAPC,PLPC, POPC, PPPC, LYSO and PAPC indicate 1-hexadecyl-2-arachidonyl-,1-stearoyl-2-arachidonyl-, 1-palmitoyl-2-linoleyl-,1-palmitoyl-2-oleyl-, 1-palmitoyl-2-palmitoyl-, 1-palmitoyl-,1-palmitoyl-2-arachidonyl-phosphatidylcholine, respectively. PAPE andSAPI indicate 1-palmitoyl-2-arachidonyl-phosphotidylethanolamine and1-stearoyl-2-arachidonyl-phosphoinositol, respectively. In all cases the¹⁴C-labelled fatty acid is in the sn-2 position.

FIG. 7: A 4–20% SDS-PAGE of lysates (5×10¹⁰ cpm/lane) of ³⁵S-methioninelabelled COS cells transfected with, no DNA, pED (no insert), clone 9reverse orientation, clones 9, 31 and 49; lanes 1–6, respectively.Molecular weight markers are indicated.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have found surprisingly a calcium independentcytosolic phospholipase enzyme, designated calcium independent cytosolicphospholipase A₂/B or calcium independent cPLA₂/B, purified from thecytosol of Chinese hamster ovary (CHO) cells. The activity was alsopresent in the cytosol of tissues and cell extracts listed in Table I.

TABLE 1 mixed micelle pH 7 liposome pH 7 tissue/cell (pmol/min/mg)(pmol/min/mg) rat brain 1–2 rat heart 0.3–0.5 bovine brain 0.4 pig heart0.8 CHO-Dukx 10–20 2–5 U937 (ATCC CRL1593) 2 FBHE (ATCC CRL1395) 2 H9c2(ATCC Ccl 108) 15

The enzyme was originally purified by more than 8,000-fold from CHOcells by sequential chromatography on diethylaminoethane (DEAE), phenyland heparin-toyopearl, followed by chromatofocussing on Mono P (asdescribed further in Example 1). In addition the activity could befurther purified by size exclusion chromatography after the Mono Pcolumn. The enzyme eluted from the size exclusion chromatography columnin the 250–350 kD range, indicating the active enzyme may consist of amultimeric complex, or may possibly be associated with phospholipids.

The calcium independent phospholipase activity correlated with a singlemajor protein band of 86 kD on denaturing sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) of active fractions fromthe Mono P and size exclusion chromatographic steps; in the latter noprotein bands were observed in the 250–350 kD range. The specificactivity of the enzyme is about 1 μmol to about 20 μmol per minute permilligram based on the abundance of the 86 kD band in the most activefractions eluted from the Mono P and size exclusion columns in the mixedmicelle assay (Example 3B). The protein band was not recognized by apolyclonal antibody directed against the calcium dependent cPLA₂ of U.S.Pat. No. 5,322,776.

The calcium independent phospholipase of the present invention has a pHoptimum of 6; its activity is suppressed by calcium (in all assays) andby triton X-100 (in the assay of Example 3A); and is not stimulated byadenosine triphosphate (ATP) (in the assay of Example 3A). The enzyme isinactivated by high concentration denaturants, e.g. urea above 3M, andby detergents, e.g. CHAPS and octyl glucoside. The calcium-independentphospholipase favors hydrolysis by several fold of unsaturated fattyacids, e.g. linoleyl, oleyl and arachidonyl, at the sn-2 position of aphospholipid compared with palmitoyl. In addition there is a preferencefor palmitoyl at the sn-1 position over hexadecyl or stearoyl forarachidonyl hydrolysis at the sn-2 position. In terms of head groupsubstituents there is a clear preference for inositol over choline orethanolamine when arachidonyl is being hydrolyzed at the sn-2 position.Further, as with cPLA₂ of U.S. Pat. No. 5,322,776, there is asignificant lysophospholipase activity, i.e. hydrolysis of palmitoyl atthe sn-1 position when there is no fatty acid substituent at the sn-2position. Finally, hydrolysis of fatty acid substituents in the sn-1 orsn-2 in PAPC were compared where either palmitoyl or arachidonyl werelabelled with ¹⁴C. Fatty acids were removed at both positions with thesn-2 position having a higher initial rate of hydrolysis by 2–3 fold.This result may indicate sequential hydrolysis of the arachidonylsubstituent followed by rapid cleavage of palmitoyl in thelysophospholipid species, which is suggested by the hydrolysis of theindividual lipid species. The similar rates of hydrolysis of fatty acidsubstituents at the sn-1 (palmitoyl) or sn-2 (arachidonyl) positions,where the radioactive label is in either position, is indicative of aphospholipase B activity. However, the fatty acid substituent at thesn-2 position clearly influences the PLB activity, not the sn-1 fattyacid, since hydrolysis of 1,2-dipalmitoyl substituted phospholipids issubstantially less than for the 1-palmitoyl-2-arachidonyl species. Theseresults can be clarified by studying the hydrolysis rates at eachposition of isotopically dual labelled phospholipids, e.g. ³H and ¹⁴Ccontaining fatty acids at the sn-1 and sn-2 positions, respectively.Therefore, it is prudent to designate the enzyme as a phospholipaseA₂/B.

A cDNA encoding the calcium independent cPLA₂/B of the present inventionwas isolated as described in Example 4. The sequence of the cDNA isreported as SEQ ID NO:1. The amino acid sequence encoded by such cDNA isSEQ ID NO:2. The invention also encompasses allelic variations of thecDNA sequence as set forth in SEQ ID NO:1, that is, naturally-occurringalternative forms of the cDNA of SEQ ID NO: 1 which also encodephospholipase enzymes of the present invention.

Other cDNAs encoding a calcium independent cPLA₂/B of the presentinvention were isolated from human cDNA sources. Two clones identifiedas “19 a” and “19 b” were isolated from a Raji cell DNA library derivedfrom Burkitt's lymphoma (ATCC CCL86, commercially available fromClontech) using a probe derived from the CHO sequence (a 2.1 kbSalI-SmaI fragment). Clones 19 a and 19 b were deposited with theAmerican Type Culture Collection on Nov. 7, 1995 as accession numbersATCC 69948 and ATCC 69949. The nucleotide sequences of clones 19 a and19 b are reported in SEQ ID NO:16 and SEQ ID NO:18, respectively. SEQ IDNO:17 and SEQ ID NO:19 report the corresponding amino acid sequencesencoded by the coding regions of clones 19 a and 19 b, respectively.Clones 19 a and 19 b are both partial clones human enzyme.

SEQ ID NO:20 and SEQ ID NO:22 report the nucleotide seqeunces ofalternative ways in which clones 19 a and 19 b can be spliced to encodea longer partial clone for the full-length human enzyme. The spliceoccurs after nucleotide 1225 in SEQ ID NO:20 and after nucleotide 1228in SEQ ID NO:22. The corresponding spliced amino acid sequences arereported in SEQ ID NO:21 and SEQ ID NO:23. Spliced cDNA clones can bemade from clones 19 a and 19 b in accordance with methods known to thoseskilled in the art.

Full-length clones encoding the human enzyme can be isolated by probinghuman cDNA libraries containing full-length clones using probes derivedfrom SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22.

Also included in the invention are isolated DNAs which hybridize to theDNA sequence set forth in SEQ ID NO:1, SEQ ID NO:16, SEQ ID NO:18, SEQID NO:20 or SEQ ID NO:22 under stringent (e.g. 4×SSC at 65° C. or 50%formamide and 4×SSC at 42° C.), or relaxed (4×SSC at 50° C. or 30–40%formamide at 42° C.) conditions.

The isolated polynucleotides of the invention may be operably linked toan expression control sequence such as the pMT2 or pED expressionvectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485–4490(1991), in order to produce the phospholipase enzyme peptidesrecombinantly. Many suitable expression control sequences are known inthe art. General methods of expressing recombinant proteins are alsoknown and are exemplified in R. Kaufman, Methods in Enzymology 185,537–566 (1990). As defined herein “operably linked” means enzymaticallyor chemically ligated to form a covalent bond between the isolatedpolynucleotide of the invention and the expression control sequence, insuch a way that the phospholipase enzyme peptide is expressed by a hostcell which has been transformed (transfected) with the ligatedpolynucleotide/expression control sequence.

A number of types of cells may act as suitable host cells for expressionof the phospholipase enzyme peptide. Suitable host cells are capable ofattaching carbohydrate side chains characteristic of functionalphospholipase enzyme peptide. Such capability may arise by virtue of thepresence of a suitable glycosylating enzyme within the host cell,whether naturally occurring, induced by chemical mutagenesis, or throughtransfection of the host cell with a suitable expression plasmidcontaining a polynucleotide encoding the glycosylating enzyme. Hostcells include, for example, monkey COS cells, Chinese Hamster Ovary(CHO) cells, human kidney 293 cells, human epidermal A431 cells, humanColo205 cells, 3T3 cells, CV-1 cells, other transformed primate celllines, normal diploid cells, cell strains derived from in vitro cultureof primary tissue, primary explants, HeLa cells, mouse L cells, BHK,HL-60, U937, or Hak cells.

The phospholipase enzyme peptide may also be produced by operablylinking the isolated polynucleotide of the invention to suitable controlsequences in one or more insect expression vectors, and employing aninsect expression system. Materials and methods for baculovirus/insectcell expression systems are commercially available in kit form from,e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBac® kit), and suchmethods are well known in the art, as described in Summers and Smith,Texas Agricultural Experiment Station Bulletin No. 1555 (1987),incorporated herein by reference.

Alternatively, it may be possible to produce the phospholipase enzymepeptide in lower eukaryotes such as yeast or in prokaryotes such asbacteria. Potentially suitable yeast strains include Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains. Candida,or any yeast strain capable of expressing heterologous proteins.Potentially suitable bacterial strains include Escherichia coli,Bacillus subtilis, Salmonella typhimurium, or any bacterial straincapable of expressing heterologous proteins. If the phospholipase enzymepeptide is made in yeast or bacteria, it is necessary to attach theappropriate carbohydrates to the appropriate sites on the protein moietycovalently, in order to obtain the glycosylated phospholipase enzymepeptide. Such covalent attachments may be accomplished using knownchemical or enzymatic methods.

The phospholipase enzyme peptide of the invention may also be expressedas a product of transgenic animals, e.g., as a component of the milk oftransgenic cows, goats, pigs, or sheep which are characterized bysomatic or germ cells containing a polynucleotide encoding thephospholipase enzyme peptide.

The phospholipase enzyme peptide of the invention may be prepared byculturing transformed host cells under culture conditions necessary toexpress a phospholipase enzyme peptide of the present invention. Theresulting expressed protein may then be purified from culture medium orcell extracts as described in the examples below.

Alternatively, the phospholipase enzyme peptide of the invention isconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. Following the concentration step, the concentrate can be appliedto a purification matrix such as a gel filtration medium. Alternatively,an anion exchange resin can be employed, for example, a matrix orsubstrate having pendant diethylaminoethyl (DEAE) groups. The matricescan be acrylamide, agarose, dextran, cellulose or other types commonlyemployed in protein purification. Alternatively, a cation exchange stepcan be employed. Suitable cation exchangers include various insolublematrices comprising sulfopropyl or carboxymethyl groups. Sulfopropylgroups are preferred (e.g., S-Sepharose® columns). The purification ofthe phospholipase enzyme peptide from culture supernatant may alsoinclude one or more column steps over such affinity resins asconcanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GASepharose®; or by hydrophobic interaction chromatography using suchresins as phenyl ether, butyl ether, or propyl ether; or byimmunoaffinity chromatography.

Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify the phospholipase enzyme peptide. Some or allof the foregoing purification steps, in various combinations, can alsobe employed to provide a substantially homogeneous isolated recombinantprotein. The phospholipase enzyme peptide thus purified is substantiallyfree of other mammalian proteins and is defined in accordance with thepresent invention as “isolated phospholipase enzyme peptide”.

The calcium independent cPLA₂/B of the present invention is distinctfrom the cPLA₂ of U.S. Pat. No. 5,322,776 and from previously-describedcalcium independent phospholipase A₂ enzymes (such as those described byGross et al., supra; and Ackermann et al., supra). The enzyme of thepresent invention differs from the cPLA₂ of the '776 patent in thefollowing ways:

-   -   (1) its activity is not calcium dependent;    -   (2) it is more active in 10% glycerol than in 70% glycerol;    -   (3) it has a molecular weight of 86 kD, not 110 kD as for cPLA₂;    -   (4) it has a pH optimum of 6, not greater than 8 as for cPLA₂;    -   (5) it hydrolyzes fatty acids at sn-1 as well as sn-2;    -   (6) it binds to heparin, while cPLA₂ does not;    -   (7) it elutes from an anion exchange column at 0.1–0.2 M NaCl,        while cPLA₂ elutes at 0.3-0.4 M NaCl; and    -   (8) it does not bind to anti-cPLA₂ polyclonal antibody

The enzyme of the present invention differs from the calcium independentenzyme of Gross et al. in the following characteristics:

-   -   (1) it has a molecular weight of 86 kD, not 40 kD as for the        Gross enzyme;    -   (2) it is not homologous at the protein level to rabbit skeletal        muscle phosphofructokinase in contrast to the 85 kD putative        regulatory protein associated with the 40 kD Gross enzyme;    -   (3) hydrolysis at the sn-2 position is favored by an acyl-linked        fatty acid at the sn-1 position in contrast to ether-linked        fatty acids with the Gross enzyme;    -   (4) its does not bind to an ATP column and was not activated by        ATP in a liposome assay compared to the Gross enzyme; and    -   (5) it was active in a mixed micelle assay containing Triton        X-100.

The enzyme of the present invention differs from the calcium independentenzyme of Ackermann et al. (the “Dennis enzyme”) in the followingcharacteristics:

-   -   (1) it does not bind to an ATP column;    -   (2) it binds to an anion exchange column (mono Q), while the        Dennis enzyme remains in the unbound fraction;    -   (3) it has a molecular weight of 86 kD, not 74 kD as for the        Dennis enzyme;    -   (4) it has substantial lysophospholipase activity and is        relatively inactive on phospholipids containing ether-linked        fatty acids at the sn-1 position in a liposome assay; and    -   (5) it appears to hydrolyze fatty acid substituents at the sn-1        and sn-2 positions of a phospholipid, whereas the Dennis enzyme        favors hydrolysis at the sn-2 position.

The calcium independent cPLA₂/B of the present invention may be used toscreen unknown compounds having anti-inflammatory activity mediated bythe various components of the arachidonic acid cascade. Many assays forphospholipase activity are known and may be used with the calciumindependent phospholipase A₂/B on the present invention to screenunknown compounds. For example, such an assay may be a mixed micelleassay as described in Example 3. Other known phospholipase activityassays include, without limitation, those disclosed in U.S. Pat. No.5,322,776. These assays may be performed manually or may be automated orrobotized for faster screening. Methods of automation and robotizationare known to those skilled in the art.

In one possible screening assay, a first mixture is formed by combininga phospholipase enzyme peptide of the present invention with aphospholipid cleavable by such peptide, and the amount of hydrolysis inthe first mixture (B₀) is measured. A second mixture is also formed bycombining the peptide, the phospholipid and the compound or agent to bescreened, and the amount of hydrolysis in the second mixture (B) ismeasured. The amounts of hydrolysis in the first and second mixtures arecompared, for example, by performing a B/B_(o) calculation. A compoundor agent is considered to be capable of inhibiting phospholipaseactivity (i.e., providing anti-inflammatory activity) if a decrease inhydrolysis in the second mixture as compared to the first mixture isobserved. The formulation and optimization of mixtures is within thelevel of skill in the art, such mixtures may also contain buffers andsalts necessary to enhance or to optimize the assay, and additionalcontrol assays may be included in the screening assay of the invention.

Other uses for the calcium independent cPLA₂/B of the present inventionare in the development of monoclonal and polyclonal antibodies. Suchantibodies may be generated by employing purified forms of the calciumindependent cPLA₂ or immunogenic fragments thereof as an antigen usingstandard methods for the development of polyclonal and monoclonalantibodies as are known to those skilled in the art. Such polyclonal ormonoclonal antibodies are useful as research or diagnostic tools, andfurther may be used to study phospholipase A₂ activity and inflammatoryconditions.

Pharmaceutical compositions containing anti-inflammatory agents (i.e.,inhibitors) identified by the screening method of the present inventionmay be employed to treat, for example, a number of inflammatoryconditions such as rheumatoid arthritis, psoriasis, asthma, inflammatorybowel disease and other diseases mediated by increased levels ofprostaglandins, leukotriene, or platelet activating factor.Pharmaceutical compositions of the invention comprise a therapeuticallyeffective amount of a calcium independent cPLA₂ inhibitor compound firstidentified according to the present invention in a mixture with anoptional pharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredient(s).The term “therapeutically effective amount” means the total amount ofeach active component of the method or composition that is sufficient toshow a meaningful patient benefit, i.e., healing or amelioration ofchronic conditions or increase in rate of healing or amelioration. Whenapplied to an individual active ingredient, administered alone, the termrefers to that ingredient alone. When applied to a combination, the termrefers to combined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously. A therapeutically effective dose of the inhibitor ofthis invention is contemplated to be in the range of about 0.1 μg toabout 100 mg per kg body weight per application. It is contemplated thatthe duration of each application of the inhibitor will be in the rangeof 12 to 24 hours of continuous administration. The characteristics ofthe carrier or other material will depend on the route ofadministration.

The amount of inhibitor in the pharmaceutical composition of the presentinvention will depend upon the nature and severity of the conditionbeing treated, and on the nature of prior treatments which the patienthas undergone. Ultimately, the attending physician will decide theamount of inhibitor with which to treat each individual patient.Initially, the attending physician will administer low doses ofinhibitor and observe the patient's response. Larger doses of inhibitormay be administered until the optimal therapeutic effect is obtained forthe patient, and at that point the dosage is not increased further.

Administration is preferably intravenous, but other known methods ofadministration for anti-inflammatory agents may be used. Administrationof the anti-inflammatory compounds identified by the method of theinvention can be carried out in a variety of conventional ways. Forexample, for topical administration, the anti-inflammatory compound ofthe invention will be in the form of a pyrogen-free, dermatologicallyacceptable liquid or semi-solid formulation such as an ointment, cream,lotion, foam or gel. The preparation of such topically appliedformulations is within the skill in the art. Gel formulation shouldcontain, in addition to the anti-inflammatory compound, about 2 to about5% W/W of a gelling agent. The gelling agent may also function tostabilize the active ingredient and preferably should be water soluble.The formulation should also contain about 2% W/V of a bactericidal agentand a buffering agent. Exemplary gels include ethyl, methyl, and propylcelluloses. Preferred gels include carboxypolymethylene such as Carbopol(934P; B.F. Goodrich), hydroxypropyl methylcellulose phthalates such asMethocel (K100M premium; Merril Dow), cellulose gums such as Blanose(7HF; Aqualon, U.K.), xanthan gums such as Keltrol (TF; KelkoInternational), hydroxyethyl cellulose oxides such as Polyox (WSR 303;Union Carbide), propylene glycols, polyethylene glycols and mixturesthereof. If Carbopol is used, a neutralizing agent, such as NaOH, isalso required in order to maintain pH in the desired range of about 7 toabout 8 and most desirably at about 7.5. Exemplary preferredbactericidal agents include steryl alcohols, especially benzyl alcohol.The buffering agent can be any of those already known in the art asuseful in preparing medicinal formulations, for example 20 mM phosphatebuffer, pH 7.5.

Cutaneous or subcutaneous injection may also be employed and in thatcase the anti-inflammatory compound of the invention will be in the formof pyrogen-free, parenterally acceptable aqueous solutions. Thepreparation of such parenterally acceptable solutions, having due regardto pH, isotonicity, stability, and the like, is within the skill in theart.

Intravenous injection may be employed, wherein the anti-inflammatorycompound of the invention will be in the form of pyrogen-free,parenterally acceptable aqueous solutions. A preferred pharmaceuticalcomposition for intravenous injection should contain, in addition to theanti-inflammatory compound, an isotonic vehicle such as Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, Lactated Ringer's Injection, or other vehicle asknown in the art. The pharmaceutical composition according to thepresent invention may also contain stabilizers, preservatives, buffers,antioxidants, or other additive known to those of skill in the art.

The amount of anti-inflammatory compound in the pharmaceuticalcomposition of the present invention will depend upon the nature andseverity of the condition being treated, and on the nature of priortreatments which the patient has undergone. Ultimately, the attendingphysician will decide the amount of anti-inflammatory compound withwhich to treat each individual patient.

Anti-inflammatory compounds identified using the method of the presentinvention may be administered alone or in combination with otheranti-inflammation agents and therapies.

EXAMPLE 1 Purification of Calcium Independent cPLA₂

A) Preparation of CHO-Dukx Cytosolic Fraction

CHO cells, approximately 5×10¹¹ cells from a 250L culture, wereconcentrated by centrifugation and rinsed once with phosphate-bufferedsaline and reconcentrated. the cell slurry was frozen in liquid nitrogenand stored at −80° C. at 4×10¹¹ cells/kg of pellet. The CHO pellets wereprocessed in 0.5 kg batches by thawing the cells in 1.2 L of 20 mMimidazol pH 7.5, 0.25M sucrose, 2 mM EDTA, 2 mM EGTA, 1 μg/ml leupeptin,5 μg/ml aprotinin, 5 mM DTT and 1 mM PMSF (“Extraction Buffer”). Thecells were transferred to a Parr bomb at 4° C. and pressurized at 600psi for 5 minutes and lysed by releasing the pressure. The supernatantwas centrifuged at 10,000×g for 30 minutes and subsequently at 100,000×gfor 60 minutes.

B) DEAE Anion Exchange Chromatography

The cytosolic fraction (10 gm protein) was diluted to 5 mg/ml with 20 mMimidazol pH 7.5, 5 mM DTT, 1 mM EDTA and 1 mM EGTA (Buffer A) andapplied to a 1 L column of DEAE toyopearl equilibrated in buffer A at 16ml/min. The column was washed to background absorbance (A₂₈₀) withbuffer A and developed with a gradient of 0–0.5M NaCl in buffer A over240 minutes with one minute fractions. The first activity peak at100–150 mM NaCl was collected.

C) Hydrophobic Interaction and Heparin Toyopearl Chromatography

The DEAE fractions (4 gm of protein at 3 mg/ml) were made 0.5M inammonium sulfate and applied at 10 μl/min to a 300 ml phenyl toyopearlcolumn equilibrated in buffer A containing 0.5 M ammonium sulfate. Thecolumn was washed to background absorbance (A₂₈₀). The column was thendeveloped with a gradient of 0.5–0.2M (15 minutes) then 0.2–0.0 Mammonium sulfate (85 minutes). The column was then connected in tandemto a 10 ml heparin column equilibrated in buffer A and elution wascontinued for 18 hours at 1.5 ml/min with buffer A. The phenyl columnwas disconnected and the activity was eluted from the heparin column byapplying 0.5M NaCl in buffer A at 2 ml/min.

D) Chromatofocussing Chromatography

A portion of the above active fractions (16 mg) was dialyzedexhaustively against 20 mM Bis-Tris pH 7, 10% glycerol, 1M urea and 5 mMDTT and applied at 0.5 ml/min to a Mono P 5/20 column equilibrated withthe same buffer. The column was washed with the same buffer tobackground absorbance (A₂₈₀) and a pH gradient was established byapplying 10% polybuffer 74 pH 5, 10% glycerol, 1 M urea and 5 mM DTT.

The relative purification of the enzyme of the present invention at eachstep of the foregoing purification scheme is summarized in Table II.

TABLE II Specific Fold Protein Activity Activity Purifi- Yield Step (mg)(u**) (u/mg) cation (%) cytosolic 126,000 2050 0.016 — — extract* DEAE16,000 1264 0.079 5 60 phenyl/ 193 90 0.46 30 4.5 heparin Mono P 0.1–0.214 140 8,000 0.7 *Extract from 3.5 kg of frozen CHO cell pellet **1 unitis defined as the amount of activity that releases 1 nmol of arachidonicacid per minute

The phospholipase can be further purified by the following steps:

E) Heparin Chromatography

The sample from (D) above is applied at 0.5 ml/min onto a heparin column(maximum capacity 10 mg protein/ml of resin) equilibrated in buffer A.The activity is eluted by 0.4 M NaCl in buffer A.

F) Size Exclusion Chromatography

The active fractions from the heparin column are applied to two TSKG3000SW_(XL) columns (7.8 mm×30 cm) linked in tandem equilibrated with150 in M NaCl in buffer A at 0.3 ml/min. Phospholipase activity elutesin the 250–350 kD size range.

Recombinant enzyme may also be purified in accordance with this example.

EXAMPLE 2 Amino Acid Sequencing

A portion (63 μg total protein) of the Mono P active fractions wasconcentrated on a heparin column, as described above. The sample, 0.36ml was mixed with an equal volume of buffer A and 10% SDS, 10 μl andconcentrated to 40 μl on an Amicon-30 microconcentrator. The sample wasdiluted with buffer A, 100 μl, concentrated to 60 μl and diluted withLaemmli buffer (2x), 40 μl. The solution was boiled for 5 minutes andloaded in three aliquots on a 4–20% gradient SDS-PAGE mini gel. Thesample was electophoresed for two hours at 120 v, stained for 20 minutesin 0.2% Blue R-250, 20% methanol and 0.5% acetic acid and destained in30% methanol (Rosenfeld et. al. Anal. Biochem. 203, pp. 173–179, 1992).Briefly, the protein bands corresponding to the phospholipase wereexcised from the gel with a razor blade and washed with 4 150 μlaliquots of 200 mM NH₄HCO₃, 50% acetonitrile, for a total of 2 hours.The gel pieces were allowed to air dry for approximately 5 minutes, thenpartially rehydrated with 1 μl of 200 mM NH₄HCO₃, 0.02% Tween 20(Pierce) and 2 μl of 0.25 μg/μl trypsin (Promega). Gel slices wereplaced into the bottom of 500 μl mini-Eppendorf tubes, covered with 30μl 200 mM NH₄HCO₃, and incubated at 37 C for 15 hours. After 1–2 minutesof centrifugation in an Eppendorf microfuge, the supernatants wereremoved and saved. Peptides in the gel slices were extracted byagitation for a total of 40 minutes with 2 100 μl aliquots of 60%acetonitrile, 0.1% TFA. The extracts were combined with the previoussupernatant. The volume was reduced by lyophilization to about 150 μl,and then the sample was diluted with 750 μl 0.1% TFA. Peptide maps wererun on an ABI 130A Separation System HPLC and an ABI 30×2.1 mm RP-300column. The gradient used was as follows: 0–13.5 minutes 0% B, 13.5–63.5minutes 0–100% B and 63.5–68.5 minutes 100% B, where A is 0.1% TFA and Bis 0.085% TFA, 70% acetonitrile. Peptides were then sequenced on an ABI470A gas-phase sequencer.

EXAMPLE 3 Phopholipase Assays

1. sn-2 Hydrolysis Assays

A) Liposome

The lipid, e.g.1-palmitoyl-2-[¹⁴C]arachidonyl-sn-glycero-3-phosphocholine (PAPC), 55mCi/mmol, was dried under a stream of nitrogen and solubilized inethanol. The assay buffer contained 100 mM Tris-HCl pH 7, 4 mM EDTA, 4mM EGTA, 10% glycerol and 25 μM of labelled PAPC, where the volume ofethanol added was no more than 10% of the final assay volume. Thereaction was incubated for 30 minutes at 37° C. and quenched by theaddition of two volumes of heptane:isopropanol:0.5 M sulfuric acid(105:20:1 v/v). Half of the organic was applied to a disposable silicagel column in a vacuum manifold positioned over a scintillation vial,and the free arachidonic was eluted by the addition of ethyl ether (1ml). The level of radioactivity was measured by liquid scintillation.

Variations on this assay replace EDTA and EGTA with 10 mM CaCl₂.

B) Mixed Micelle Basic

The lipid was dried down as in (A) and to this was added the assaybuffer consisting of 80 mM glycine pH 9, 5 mM CaCl₂ or 5 mM EDTA, 10% or70% glycerol and 200 μM triton X-100. The mixture was then sonicated for30–60 seconds at 4° C. to form mixed micelles.

C) Mixed Micelle Neutral

As for (B) except 100 mM Tris-HCl pH 7 was used instead of glycine asthe buffer.

2. sn-1 Hydrolysis Assays

Sn-1 hydrolysis assays are performed as described above for sn-1hydrolysis, but using phospholipids labelled at the sn-1 substituent,e.g. 1-[¹⁴C]-palmitoyl-2-arachidonyl-sn-glycero-3-phophocholine.

EXAMPLE 4 Cloning of Calcium Independent cPLA₂/B

A) cDNA Library Construction

Total RNA was first prepared from 2×10⁸ CHO-DUX cells using the RNAgentstotal RNA kit (Promega, Madison, Wis.) and further purified using thePolyATract mRNA Isolation System (Promega) to yield 13.2 μg polyA+ mRNA.Double stranded cDNA was prepared by the Superscript Choice System(Gibco/BRL, Gaithersburg, Md.) starting with 2 μg of CHO-DUX mRNA andusing oligo dT primer. The cDNA was modified at both ends by addition ofan EcoRI adapter/linker provided by the kit. These fragments were thenligated into the predigested lambda ZAPII/EcoRI vector, and packagedinto phage particles with Gigapack Gold packaging extracts (Stratagene,La Jolla, Calif.).

B) Oligonucleotide Probe Design

Several of the peptide sequences determined for the purified calciumindependent PLA₂/B were selected to design oligonucleotide probes. Theamino acid sequence from amino acid 361 to 367 of SEQ ID NO:2 was usedto design two degenerate oligonucleotide pools of 17 residues each. Pool1 is 8-fold degenerate representing the sense strand for amino acids 361to 366 of SEQ ID NO:2, and pool 2 is 12-fold degenerate representing theantisense strand for amino acids 362–367 of SEQ ID NO:2. Two otherdegenerate pools were also made from other sequences. Pool 3 is 32-folddegenerate and represents the sense strand for amino acids 490 to 495 ofSEQ ID NO:2, and pool 4 is 64-fold degenerate representing the antisensestrand for amino acids 513 to 518 of SEQ ID NO:2.

C) Library Screening

Approximately 400,000 recombinant bacteriophage from the CHO-DUX cDNAlibrary were plated and duplicate nitrocellulose filters were prepared.One set of filters was hybridized with pool 1 and the other with pool 2using tetramethylammonium chloride buffer conditions (Jacobs et al.,Nature, 1985, 313, 806). Twelve positive bacteriophages were identifiedand plated for further analysis. Three sets of nitrocellulose filterswere prepared from this plating and hybridized with pools 2, 3 and 4, torepresent the three peptide sequences from which probes were designed.Several clones were positive for all three pools. Individualbacteriophage plaques were eluted and ampicillin resistant plasmidcolonies were prepared following the manufacturer's protocols(Stratagene). Plasmid DNA was prepared for clones 9, 17, 31 and 49, andrestriction digests revealed 3.0 kb inserts. Analysis of a portion ofthe DNA sequence in these clones confirmed that they contained severalcPLA₂/B peptide sequences and represented the complete coding region ofthe gene. Clone 9 was selected for complete DNA sequence determination.The sequence of clone 9 is reported as SEQ ID NO:1.

Clone 9 was deposited with ATCC on Jul. 27, 1994 as accession number69669.

EXAMPLE 5 Expression of Recombinant cPLA₂/B

A) Expression in COS Cells

Clone 9 from Example 4 was excised inserted into a SalI site that wasengineered into the EcoRI site of the COS expression vector, PMT-2, abeta lactamase derivative of p91023 (Wong et al., Science, 1985, 228,810). 8 μg of plasmid DNA was then transfected into 1×10⁶ COS cells in a10 cm dish by the DEAE dextran protocol (Sompayrac et al., Proc. Natl.Acad. Sci. USA, 1981, 78, 7575) with the addition of a 0.1 mMchloroquine to the transfection medium, followed by incubation for 3hours at 37° C. The cells were grown in conventional media (DME, 10%fetal calf serum). At 40–48 hours post-transfection the cells werewashed twice and then incubated at 37° C. in PBS, 1 mM EDTA (5 ml). Thecells were then collected by centrifugation, resuspended in ExtractionBuffer (0.5 ml), and lysed by 20 strokes in a Dounce at 4° C. The lysatewas clarified by centifugation and 10–50 μl of the cytosolic fractionwas assayed in the neutral and pH 9 mixed micelle assays.

In a further experiment, COS cells were transiently transfectedaccording to established procedures (Kaufman et al.). After 40–48 hourspost-tranmsfection the cells wer labelled with ³⁵S-methionine, 200 μCiper 10 cm plate, for one hour and the cells were lysed in NP-40 lysisbuffer (Kaufman et al.). The cell lysates were analyzed by SDS-PAGE on a4–20% reducing gel where equal counts were loaded per lane. There was anadditional protein band at 84–86 kD in the lysates from cellstransfected with clones 9, 31 and 49, but not in controls (see FIG. 7).

B) Expression in CHO Cells

A single plasmid bearing both the cPLA₂/B encoding sequence and a DHFRgene, or two separate plasmids bearing such sequences, are introducedinto DHFR-deficient CHO cells (such as Dukx-BII) by calcium phosphatecoprecipitation and transfection. DHFR expressing transformants areselected for growth in alpha media with dialyzed fetal calf serum.Transformants are checked for expression of recombinant enzyme bybioassay, immunoassay or RNA blotting and positive pools aresubsequently selected for amplification by growth in increasingconcentrations of methotrexate (MTX) (sequential steps in 0.02, 0.2, 1.0and 5 μM MTX) as described in Kaufman et al., Mol. Cell Biol., 1983, 5,1750. The amplified lines are cloned and recombinant enzyme expressionis monitored by the mixed micelle assay. Recombinant enzyme expressionis expected to increase with increasing levels of MTX resistance.

EXAMPLE 6 Mutagenesis of Serine Residues

Ser252 and Ser465 of the murine cPLA₂/B amino acid sequence were mutatedto alanine residues using the Chamelon Mutagenesis kit (Stratagene)using oligonucleotides CATGGGACCCGCTGGCTTTCC (SEQ ID NO:24) andGGCAGGAACCGCCACTGGGGGC (SEQ ID NO:25), respectively. PLA₂ activity wasabrogated by changing Ser465 to Ala in the lipase consensus sequence(GXSXGG) surrounding that residue. Although Ser252 is found in a partiallipase motif, mutagenesis did not result in loss of activity. Moreover,Ser465, and the lipase consensus sequence surrounding this residue, areconserved in the human sequence (see amino acids 462–467 of SEQ ID NO:21and 463–468 of SEQ ID NO:23), while Ser252 is not. On this basis, it isbelieved that this conserved serine residue is required for activity.

Patent and literature references cited herein are incorporated byreference as if fully set forth.

1. A purified antibody that specifically binds to an epitope in an aminoacid sequence selected from: (a) the amino acid sequence of SEQ IDNO:17; (b) the amino acid sequence of SEQ ID NO:19; (c) a fragment ofthe amino acid sequence of SEQ ID NO:19 having activity in a mixedmicelle assay with 1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidyleholine;(d) the amino acid sequence of SEQ ID NO:21; (e) a fragment of the aminoacid sequence of SEQ ID NO:21 having activity in a mixed micelle assaywith 1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidyicholine; (f) the aminoacid sequence of SEQ ID NO:23; and (g) a fragment of the amino acidsequence of SEQ ID NO:23 having activity in a mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidyleholine.
 2. The antibody ofclaim 1, wherein the epitope is in the amino acid sequence of SEQ IDNO:17.
 3. The antibody of claim 1, wherein the epitope is in the aminoacid sequence of SEQ ID NO:19.
 4. The antibody of claim 1, wherein theepitope is in the fragment of the amino acid sequence of SEQ ID NO: 19having activity in a mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine.
 5. The antibody ofclaim 1, wherein the epitope is in the amino acid sequence of SEQ IDNO:21.
 6. The antibody of claim 1, wherein the epitope is in thefragment of the amino acid sequence of SEQ ID NO:21 having activity in amixed micelle assay with 1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine.
 7. The antibody of claim 1, wherein the epitope is in the aminoacid sequence of SEQ ID NO:23.
 8. The antibody of claim 1, wherein theepitope is in the fragment of the amino acid sequence of SEQ ID NO:23having activity in a mixed micelle assay with1-palmitoyl-2-[¹⁴C]-arachidonyl-phosphatidylcholine.
 9. The antibody ofclaim 1, wherein the antibody is a monoclonal antibody.
 10. The antibodyof claim 1, wherein the antibody is a polyclonal antibody.
 11. Acomposition comprising the antibody of claim 1 and a pharmaceuticallyacceptable carrier.